This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Oxalate, a compound that is toxic to almost all organisms, is the primary energy source for Oxalobacter formigenes, a bacterium that is present in the gut of several mammalian species including humans. In this microorganism, oxalate is converted into formate and carbon dioxide in a catalytic cycle involving the enzymes oxalyl-CoA decarboxylase and formyl-CoA transferase (FRC). The latter enzyme is of special interest because it adopts a spectacular new fold in which the two subunits are linked together in an interlocked dimer, like two rings of a chain [1]. This fold, in fact, is characteristic for all enzymes in the Class III family of CoA transferases [2], as seen in the crystal structures of the formyl-CoA transferase ortholog in Escherichia coli coded by the yfdW gene [3], and butyrobetaine-CoA:carnitine CoA transferase [4]. As part of combined experimental and theoretical efforts to (i) understand the molecular principles that give rise to the interlocked dimer structure, and (ii) elucidate the role of a conformationally mobile tetraglycine loop (Gly258-Gly259-Gly260-Gly261) located within the active site [1], we are interested in characterizing the dynamical properties of the FRC dimer, and their modulation by substrate binding. In our initial studies, which have a key goal of obtaining sufficient preliminary data for acquiring external project funding, we seek to examine the relationship between the tetraglycine loop motions and the low-frequency normal modes of the apo-enzyme, and several FRC/substrate and FRC/intermediate complexes that we have observed by X-ray crystallography [5,6]. In preliminary experiments using the local computing facilities at the University of Florida (UF), we have explored the use of elastic network models, primarily because of their computational simplicity [7]. It is, however, very difficult to include the effects of ligand binding into such models and so we now wish to pursue more computationally demanding normal mode analyses [8]. In particular, we aim to calculate the large-scale vibrational motions of the FRC dimer using the DIMB (Diagonalization In a Mixed Basis) method [9] as implemented in the CHARMM (version c34b1 or c33b2) software package [10]. Given that this system is comprised of over 16,000 atoms (even when the solvent environment is modeled using a Generalized-Born solvation model [11]) the memory requirements of such a calculation are too demanding for performing this study on local UF high-performance computing facilities. Given these limitations in our local resources, we are requesting supercomputing time through the POPS program to perform these DIMB calculations. The results of these computational studies will be carefully evaluated by comparisons with crystallographic temperature factors [12]. The impact of substrate binding and reaction intermediates on the correlated motions of the tetraglycine loop and the side chain of Trp48 will be of particular interest in these studies as recent work in our laboratory has demonstrated the importance of this tryptophan in precluding the inhibition of FRC under conditions of high oxalate [13]. Not only will these calculations give new insights into the functional motions of the FRC dimer, they will also provide a platform for more sophisticated steered MD simulations [14] that will seek to explore the role of the tetraglycine loop in mediating catalysis. The latter set of demanding computational experiments will form the basis of a future NSF proposal submission in 2008. Literature citations 1. Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003) EMBO J. 22, 3210-3219. 2. Heider, J. (2001) FEBS Lett. 509, 345-349. 3. Gruez, A., Roig-Zamboni, V., Valencia, C., Campanacci, V., and Cambillau, C. (2003) J. Biol. Chem. 278, 34582-34586. 4. Stenmark, P., Gurmu, D., and Nordlund, P. (2004) Biochemistry 43, 13996-14003. 5. Jonsson, S., Ricagno, S., Lindqvist, Y. and Richards, N. G. J. (2004) J. Biol. Chem. 279, 36003-36012. 6. Berthold, C.L., Toyota, C.G., Richards, N.G.J., and Lindqvist, Y. (2007) J. Biol. Chem. Accepted for publication. 7. Temiz, N.A., Meirovitch, E., and Bahar, I. (2004) Proteins: Struct. Funct. Bioinf. 57, 468-480. 8. Tama, F., and Brooks, C.L., III (2006) Annu. Rev. Biophys. Biomol. Struct. 35, 115-133. 9. Mouawad, L., and Perahia, D. (1993) Biopolymers 33, 599-611. 10. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., and Karplus, M. (1983) J. Comput. Chem. 4, 187-217. 11. Bashford, D., and Case, D.A. (2000) Annu. Rev. Phys. Chem. 51, 129-152. 12. Isin, B., Doruker, P., and Bahar, I. (2002) Biophys. J. 82, 569-581. 13. Toyota, C.G., Berthold, C.L., Gruez, A., Jonsson, S., Lindqvist, Y., Cambillau, C., and Richards, N.G.J. (2007) J. Bacteriol. Submitted for publication. 14. Park, S., and Schulten, K. (2004) J. Chem. Phys. 120, 5946-5961. Work on FRC structure and mechanism has been supported by the National Institutes of Health (DK61666) and the Swedish Research Council-Scientific Council for Natural and Engineering Sciences